Centromeres Are Specialized Replication Domains in Heterochromatin
Kami Ahmad and Steven Henikoff
Howard Hughes Medical Institute, and Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
Abstract. The properties that define centromeres in complex eukaryotes are poorly understood because the underlying DNA is normally repetitive and indistinguishable
from surrounding noncentromeric sequences. However,
centromeric chromatin contains variant H3-like histones
that may specify centromeric regions. Nucleosomes are
normally assembled during DNA replication; therefore,
we examined replication and chromatin assembly at
centromeres in Drosophila cells. DNA in pericentric
heterochromatin replicates late in S phase, and so centromeres are also thought to replicate late. In contrast
to expectation, we show that centromeres replicate as
isolated domains early in S phase. These domains do not
appear to assemble conventional H3-containing nucleosomes, and deposition of the Cid centromeric H3-like
variant proceeds by a replication-independent pathway.
We suggest that late-replicating pericentric heterochromatin helps to maintain embedded centromeres
by blocking conventional nucleosome assembly early in
S phase, thereby allowing the deposition of centromeric
histones.
Key words: centromere • heterochromatin • DNA
replication • histone • Drosophila
Introduction
Centromeres are the attachment points of chromosomes
to the mitotic and meiotic spindles. Centromere activity in
the budding yeast Saccharomyces is conferred by a specific
DNA sequence (Clarke, 1990). However, the centromeres
of chromosomes in complex eukaryotes are not defined by
any distinct sequence: they are usually composed of highly
repetitive satellite sequences that are indistinguishable
from surrounding noncentromeric satellite blocks (Csink
and Henikoff, 1998; Murphy and Karpen, 1998; Willard,
1998). In Drosophila, a centromere was mapped to a 420kb region that carries full centromeric function, and which
is composed of repetitive sequences that are found at noncentromeric regions of chromosomes (Murphy and Karpen,
1995; Sun et al., 1997). Minimal centromeres in mammalian
cells are also hundreds of kilobases in size, and entirely composed of repetitive alphoid sequences (Lo et al., 1999; Yang
et al., 2000). Because centromeres cannot be delineated from
their flanking sequences by any sequence criteria, and analyzing highly repetitive DNA is difficult, the size and location of
centromeres in most organisms remains unknown. For example, molecular mapping of functional centromeres in Arabidopsis could only map the centromeres to within the extensive repetitive regions of chromosomes that includes
megabases of canonical 180-bp repeats, clusters of transposons, and even functional genes (Copenhaver et al., 1999).
Highly repetitive regions of chromosomes adopt a heterochromatic chromatin structure, with distinctive properties
and chromatin components (Elgin, 1996). Centromeres
have a different set of chromatin components, of which
centromeric nucleosomes containing special histone H3like proteins are most conspicuous. Nucleosomes are
normally assembled during replication, and it has been
suggested that the centromeric histone CENP-A is incorporated at the centromere at the time of its replication
(Shelby et al., 1997). Heterochromatin replicates late
(Lima-de-Faria and Jaworska, 1968), and so centromeres
are also thought to replicate late. Very late replication of centromeres has been proposed to play a role in centromere
function (Dupraw, 1968; Csink and Henikoff, 1998). In
contrast to expectation, we show that centromeres replicate as isolated domains early in S phase. At this time,
they are surrounded by heterochromatin that has not yet
replicated. Therefore, a fundamental feature of DNA,
replication timing, distinguishes centromeres from surrounding heterochromatin. We further show that nucleosome assembly using histone H3 is inhibited as centromeres replicate.
We suggest that pericentromeric heterochromatin sequesters
centromeres away from histone H3 and thereby participates
in centromere maintenance.
Materials and Methods
Cell Culture and Immunostaining
Address correspondence to Steven Henikoff, Howard Hughes Medical Institute, and Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A1-162, Seattle, WA 98109. Tel.: (206) 667-4515. Fax: (206)
667-5889. E-mail: [email protected]
All experiments were conducted using a Drosophila tetraploid Kc cell
line. Culturing, transfection methods, and constructs were previously described (Henikoff et al., 2000), except that the colcemid treatment in cytological preparations was omitted. To label replicating DNA with nucleotide triphosphate analogues, we administered a 15-min hypotonic
 The Rockefeller University Press, 0021-9525/2001/04/101/9 $5.00
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treatment using KHB buffer containing 0.1 mM digoxigenin (dig)1-dUTP
(Boehringer; Koberna et al., 1999), and then returning cells to insect media for 15 min. For dual-pulse labeling, cells were treated with 0.1 mM digdUTP/KHB, resuspended in insect cell culture media for the chase interval, and then treated with 0.1 mM biotin (Bio)-dUTP/KHB (Boehringer).
Labeling of replicating DNA with 5-bromodeoxyuridine was performed
by incubating cells in culture medium supplemented with 10 ␮g/ml BrdU
and 10 ␮g/ml deoxycytidine for 1 h. Immunological detection of Cid was performed on immobilized cells, which were then fixed with methanol/acetic
acid/H2O (11:11:1). Digestion with exonuclease III (30 U in 100 ␮l buffer)
was used to expose the BrdU epitope. BrdU was detected using a FITC-conjugated monoclonal anti–BrdU antibody (Boehringer), and dig-dUTP was
detected using FITC- or Texas red–conjugated anti–dig antibodies (Jackson
ImmunoResearch Laboratories). In dual-pulse experiments, the two nucleotide analogues were detected using FITC-conjugated anti–dig antibodies
and Texas red–conjugated streptavidin (Pierce Chemical Co.).
Transfection efficiencies with histone-green fluorescent protein (GFP)
constructs were typically 35–70%. To assay the localization of histoneGFP fusion proteins when replication was blocked, we split one transfected culture into a control and an experimental sample. Aphidicolin was
added to the experimental culture at a final concentration of 0.1 mg/ml 5
min before heat shock induction. Cells were allowed to recover after induction for 2 h at 25⬚C.
HP1 or Cid proteins were detected using anti–HP1 rabbit antibodies
(Smothers and Henikoff, 2001) or by using anti–Cid rabbit antibodies
(Henikoff et al., 2000) followed by anti–rabbit IgG Cy5-conjugated goat
antibodies (Jackson ImmunoResearch Laboratories). All images were
collected as previously described (Henikoff et al., 2000).
Image Quantitation
Images were analyzed using DeltaVision software (Applied Precision). Cells
were categorized according to their overall pattern of replication (euchromatic, scattered, or heterochromatic). Centromeres and nuclei were identified by thresholding anti–Cid signals and DAPI fluorescence, respectively.
To measure the amount of nucleotide incorporated in centromeres, we
examined all optical sections that included an anti–Cid signal. We selected
nuclei in which euchromatin was labeled, summed the intensity of the nucleotide analogue signal in an area defined by Cid signals, and divided this
by the summed nucleotide signal intensity within the euchromatin of the
same section. Both sums were corrected for background. The summed signal intensity indicates the amount of replication in a cell during the labeling
period, and the average ratio from randomly selected early S phase cells
(n ⫽ 22) is an estimate of the fraction of replication at centromeric foci relative to euchromatin. These integrated intensities allow an estimate of the
amount of DNA replicated early in S phase at a centromeric replication focus. The amount of nucleotide analogue incorporated at centromeric replication foci above background relative to that incorporated in euchromatin
throughout the early S phase period was 0.005 (SEM ⫽ 0.002). Since euchromatin contains ⵑ120,000 kb of DNA (Adams et al., 2000), the average
centromeric replication focus contains ⵑ500 kb of DNA.
The Hsp70 promoter has very low-level constitutive activity, so in all
cases we determined the mean GFP signal from uninduced control samples and removed from the analysis any nuclei in which the GFP signal
was within two standard deviations of this mean. We counted the number
of nuclei with appropriate GFP signals (Cid-GFP localized to centromeres
or H3GFP labeling replicating DNA), and scaled these numbers to the
transfection efficiency (estimated as the number of nuclei showing localization after induction and with no aphidicolin treatment). The amount of
histone-GFP fusion proteins incorporated at centromeres when produced
from cid promoter constructs was estimated by comparing the integrated
intensities from GFP in euchromatin to that in centromeres (n ⫽ 23 for
cid-H2BGFP, n ⫽ 10 for cid-H3GFP).
uniform and they can be easily manipulated. We used
Drosophila Kc cells, which have been used in previous
studies of replication timing, and have been documented as
having a stable karyotype (Echalier, 1997). In Kc nuclei,
there is a clear separation between the early replication of
euchromatin and the late replication of heterochromatin
during the ⵑ10-h-long S phase (Barigozzi et al., 1966; Dolfini et al., 1970). Furthermore, the heterochromatic regions
of chromosomes coalesce into a large irregular chromocenter that occupies ⵑ25% of the nuclear volume and
can be visualized by detection of the HP-1 protein (van
Steensel and Henikoff, 2000; Fig. 1, A and B). Centromeres
are embedded within the chromocenter (van Steensel and
Henikoff, 2000). A chromocenter is not unique to the Kc
nucleus, as we have observed consistent chromocenter formation in other dividing cells as well, including S2 and S3
cell lines (Ahmad, K., unpublished results). This chromocentric organization of the nucleus and the regularity of
the cell cycle allowed us to precisely follow replication timing of centromeres in asynchronous Kc cell populations.
Pulse-labeling of Kc cells with the nucleotide analogue
dig-dUTP confirms earlier studies of mitotic replication
banding (Barigozzi et al., 1966; Dolfini et al., 1970) that
heterochromatin and euchromatin replicate in distinct portions of S phase (Fig. 1, A and B). A typical experiment
showed that 51% of nuclei were in S phase at the time of
the pulse: 23% showed euchromatic replication patterns,
25% showed heterochromatic replication patterns, and 3%
showed discrete foci scattered throughout both regions.
This last type indicates that there is only minor overlap of
the euchromatic and heterochromatic replication periods
during the pulse. Euchromatin replicates in the first portion of S phase (early S), and heterochromatin in the last
(late S) (Barigozzi et al., 1966; Dolfini et al., 1970). This
spatial and temporal separation of replication in Kc cells
facilitates a cytological analysis of centromere replication.
Centromeres Replicate in Early S Phase
1
Abbreviations used in this paper: Bio, biotin; dig, digoxigenin; GFP, green
fluorescent protein.
We have previously described the centromere identifier
(cid) gene in Drosophila, which encodes a centromeric histone H3-like protein (Henikoff et al., 2000). This protein is
analogous to CENP-A in mammals (Palmer et al., 1991),
Cse4p in Saccharomyces (Stoler et al., 1995), HCP-3 in
nematodes (Buchwitz et al., 1999), and SpCenpA in
Schizosaccharomyces (Takahashi et al., 2000). All of these
proteins are thought to be assembled into specialized nucleosomes, although direct evidence has only been obtained for CENP-A (Palmer et al., 1987; Yoda et al., 2000).
Because Cid appears to be a constitutive centromeric component, antibodies to Cid can be used to locate centromeres in interphase nuclei, and typically detect four to
six centromeric spots (5.6 ⫾ 2.1) in both diploid and tetraploid Kc cells (Henikoff et al., 2000). Chromosomes in
Drosophila somatic cells are paired; therefore, each centromeric spot probably represents a cluster of centromeres
from homologous chromosomes, regardless of their total
number. These spots divide into individual centromeres
only in late-G2 phase of the cell cycle (data not shown).
We used anti–Cid antibodies on nuclei that had been
pulse labeled with nucleotide analogues to determine
when centromeric DNA replicates. The Cid epitope and
nucleotide analogues colocalize when euchromatin is also
The Journal of Cell Biology, Volume 153, 2001
102
Results
Replication of Euchromatin and Heterochromatin
in Kc Cells
Immortal cell lines are traditionally used to examine replication timing in higher eukaryotes, because cell types are
Figure 1. Euchromatin and heterochromatin replicate at distinct
times in Kc nuclei. Newly replicated DNA was visualized by incorporation of dig-dUTP (A–C)
or BrdU (D), and nuclei were
counterstained with DAPI. (A
and B) Detection with anti–HP1
antibodies stains the chromocenter; the remainder of each
nucleus is euchromatin. (A) Euchromatic replication; (B) heterochromatic replication; (C and
D) detection of centromeres by
anti–Cid antibody. In the merged
images, protein localization is in
red and sites of replicating DNA
are in green. Nuclei show on average 5.6 (⫾2.1, n ⫽ 254) centromeric spots throughout most of
the cell cycle. Incorporation of
the DNA label reveals that centromeres replicate with euchromatin. Single optical sections are
shown (z ⫽ 0.2 ␮m).
replicating, revealing that centromeres replicate in early S
phase (Fig. 1 C). Centromeres appear to be the only sites
in the chromocenter to replicate at this time. Of 42 nuclei
in which euchromatin was labeled, 20 also showed labeling
at centromeres, indicating that centromeres are replicating
through about one half of early S phase. However, within
that period, centromeres must replicate asynchronously,
since we observed incorporation at only a subset of centromeric spots in any one nucleus. Asynchronous replication
of centromeric satellite has been observed in human cells
(O’Keefe et al., 1992).
The nucleotide triphosphate pulse labeling procedure
that we used allows for detection of incorporation without
requiring DNA denaturation, thus providing excellent cytology. The brief hypotonic shock required to deliver nucleotide triphosphate analogues was found to have little or
no ill effects on a wide variety of cell processes (Koberna
et al., 1999). To rule out the possibility that the hypotonic
treatment used to deliver nucleotide triphosphate analogues had affected DNA replication, we performed the
incorporation using BrdU, which penetrates cells without
hypotonic treatment. Identical results were obtained (Fig.
1 D). We conclude that replication at centromeres initiates
substantially before that of the rest of the DNA in the heterochromatic compartment of the nucleus.
We determined the relative timing of replication in different regions in a nucleus using two sequential pulses of nu-
cleotide analogues. A pulse of dig-dUTP nucleotide analogue was administered, and this was followed by a chase
in regular media and a second pulse of Bio-dUTP. Nuclei
that had been labeled with a chase interval of 1, 2, or 3 h
generally showed clear distinction between sites of digand Bio-dUTP incorporation, consistent with the completion of euchromatic replication in individual foci within
these time intervals (Fig. 2, A and B; Manders et al., 1992).
We categorized cells according to their overall pattern of
nucleotide incorporation from these two pulses (Fig. 2 E:
E/E, euchromatin/euchromatin; E/H, euchromatin/heterochromatin; or H/H, heterochromatin/heterochromatin),
and then assessed incorporation at centromeres in each
category. Some cells with an E/E pattern showed no incorporation of dig-dUTP at centromeres, but did show centromeric incorporation of the second nucleotide BiodUTP (Fig. 2, A and E). Incorporation of dig-dUTP but
not Bio-dUTP at the centromeres of E/E pattern nuclei
was never observed (n ⫽ 125). These results demonstrate
that replication begins at euchromatic sites before centromeric origins fire. We also observed nuclei with an E/E labeling pattern in which both nucleotide analogues were incorporated into the same centromeric site, suggesting that
this site has been replicating for the entire 1-h interval between the two pulses (Fig. 2 E, right-most column). The
frequency of centromeres labeled with both nucleotide analogues suggests that replication continues for 2–3 h during the early S-phase period. Experiments in which the
two nucleotide analogues were delivered 2 and 3 h apart
Ahmad and Henikoff Centromeres Are Specialized Replication Domains
103
Prolonged Replication of Centromeres
Figure 2. Prolonged replication of centromeres. Cells were pulse labeled with
dig-dUTP followed by a second pulse 3 h later with Bio-dUTP. Anti–Cid antibodies mark the positions of the centromeres. DAPI staining is white. In the
merged images, Cid localization is in red, dig-dUTP in green, and Bio-dUTP in
blue. Coincidence of dig-dUTP and Cid is yellow, and Bio-dUTP and Cid is
magenta in the merged images. (A) An early S phase nucleus in which replication began in euchromatin before beginning in centromeres (incorporation of
the second analogue only). (B) Colocalization of both nucleotide analogues at
centromeres, indicating that some centromeres replicate for the entire period
between the two pulses. (Insets) Two centromeres from this nucleus that display incorporation of both nucleotide analogues. (C) Labeling of euchromatin and centromeres with first pulse, and the chromocenter
with the second pulse. (D) Labeling of distinct subsets of heterochromatin in the chromocenter by the two pulses, but not of centromeres. All images are single optical sections. (E) Number of centromeres labeled by nucleotide analogues. A dig-dUTP nucleotide
pulse was delivered to cells and followed by a second pulse of Bio-dUTP 1 h later. Nuclei that had incorporated both nucleotide analogues were categorized by the overall replication patterns shown by each pulse (E/E, euchromatic/euchromatic; E/H, euchromatic/
heterochromatic; H/H, heterochromatic/heterochromatic labeling). At least 10 nuclei of each combined pattern were examined. The
“heterochromatin/euchromatin” pattern was never observed. Centromeres were scored for the incorporation of each nucleotide analogue (⫺ or ⫹), and the numbers of centromeres showing incorporation of either or both analogues are shown.
confirmed this calculation, because occasional nuclei were
seen in which both dig- and Bio-dUTP were incorporated
at a single centromeric spot (Fig. 2 B). This is an unusually
prolonged period of replication since most sites complete
replication in 40–60 min (Fig. 2; Manders et al., 1992; Ma
et al., 1998). Based on the integrated intensity of nucleotide incorporation at centromeric replication foci (see
Materials and Methods), we estimate that ⵑ500 kb of
DNA at each centromere within a centromeric replication
focus is replicated in the early S-phase period.
The detection of early replication at centromeres is
aided by their clear spatial separation from simultaneously
replicating euchromatin. In nuclei with heterochromatic
labeling patterns, many replication foci are near centromeres (Fig. 2, C and D), and the high density of
foci might obscure centromere replication. To examine
whether any replication occurs at centromeres in the late S
period, we quantitated the amount of incorporated nucle-
otide analogue in centromeric spots in the early and late
periods. These measurements show that ⵑ90% of all detectable nucleotide incorporation at centromeres occurs in
the early period (316 U mean intensity/pixel per centromere in the early period versus 34 U in the late period).
Thus, the upper limit on the amount of replication at centromeres that occurs in the late S period is ⵑ10%, but
even this may be due to nearby replication foci that could
not be distinguished from centromeres. We conclude that
centromeric replication is largely complete before the rest
of the heterochromatic chromocenter replicates.
The Journal of Cell Biology, Volume 153, 2001
104
Histone H3 Is Excluded from Centromeres
Models for centromere specification by unique DNA sequences are ruled out by the absence of centromere-specific sequences in natural centromeres (Karpen and
Allshire, 1997), and the establishment of neocentromeres
Figure 3. Localization of histone-GFP fusion
proteins. (A and B) Expression of histones H2BGFP and H3-GFP from the cid promoter, which
is active early in S phase. DAPI staining is white.
In the merged images, Cid localization is in red
and histone-GFP in green. H2B-GFP localizes to
euchromatin and to centromeres (A), but H3GFP protein localizes only to euchromatin (B).
For each histone-GFP fusion, we normalized the
fluorescence intensity of GFP at centromeres to
that in euchromatin in individual nuclei. The ratio of incorporation of H2B-GFP at centromeres
relative to euchromatin was 0.016 (SEM ⫽ 0.01,
n ⫽ 23), while for H3-GFP it was 0.0014 (SEM ⫽
0.0004, n ⫽ 10). These ratios are significantly different when compared by a Mann-Whitney test
(P ⫽ 0.0002). (C and D) Expression of H2BGFP and H3-GFP, respectively, after induction
of a heat-shock promoter. Both histone-GFP fusion proteins give similar labeling of heterochromatin in late S-phase cells. Single optical sections
are shown.
at previously noncentromeric regions (Warburton et al.,
2000). An attractive alternative to DNA sequence–based
specification of centromeres is that centromeric nucleosomes define the centromere. In all eukaryotes, centromeres are likely to be contained in specialized nucleosomes that include an H3-like protein (Tyler-Smith and
Floridia, 2000). We have previously shown that Cid-GFP
fusion protein produced from the cid promoter localizes to
centromeres, and that this promoter is active early in S
phase (Henikoff et al., 2000). Thus, the normal localization of Cid takes place as centromeric DNA is replicating.
However, the deposition of specialized nucleosomes at
replicating centromeres must be difficult because histone
H3 is in vast excess throughout S phase (Osley, 1991).
We wondered whether the deposition of Cid in centromeric replication domains might be facilitated by preventing the deposition of histone H3. Conventional nucleosomes are normally assembled during replication;
therefore, we examined the deposition of conventional histones fused to GFP when centromeres replicate. Previous
work has shown that histone-GFP fusion proteins can localize to chromatin (Kanda et al., 1998; Henikoff et al.,
2000). We had previously characterized the deposition of
H2B-GFP and H3-GFP fusion proteins by examining mitotic chromosomes. When expressed from the cid promoter, which is active early in S phase, these fusion proteins localize to the euchromatic arms of chromosomes
(Henikoff et al., 2000). These experiments did not address
whether small quantities of the fusion proteins were also
incorporated at centromeres. Therefore, we examined the
deposition of histone-GFP fusion proteins after production from transfected cid-promoter constructs in interphase nuclei to determine whether these histones were incorporated in centromeric replication domains. While
The centromeric histone CENP-A can form nucleosomal
particles in vitro (Yoda et al., 2000), and cofractionates
with other histones in vivo (Palmer et al., 1987; Shelby et
al., 1997). The similarity of Drosophila Cid to histone H3
suggests that Cid is also incorporated into nucleosomes.
Since the deposition of histone H3 is inhibited as centromeres replicate, Cid-containing nucleosomes might be
formed using an alternate pathway. To investigate the nature of a Cid deposition pathway, we tested the dependence of histone-GFP protein localization on DNA replication. A pulse of histone-GFP fusion proteins can be
produced in cells by transfecting with a heat-shock promoter construct, and then inducing the promoter. Newly
produced H2B-GFP and H3-GFP proteins localize in subnuclear patterns similar to patterns produced by pulses of
Ahmad and Henikoff Centromeres Are Specialized Replication Domains
105
H2B-GFP was readily deposited at centromeres, H3-GFP
was not (P ⫽ 0.0002; Fig. 3, A and B). Both histone-GFP
proteins give similar intense labeling in euchromatin, implying that the lack of H3-GFP at centromeres is not due
to a general reduced deposition of H3-GFP. Labeling in
heterochromatin by H3-GFP and H2B-GFP produced
from heat-shock promoter constructs was also indistinguishable in intensity and pattern (Fig. 3, C and D). That
the centromere may be deficient of histone H3 has been
previously suggested, based on the absence of phosphorylated H3 antibody labeling (van Hooser et al., 1999), and
our results with H3-GFP fusion protein support this idea.
We conclude that the deposition of H3-containing nucleosomes is inhibited at the time that the cid promoter is active in early S phase.
Cid-containing Nucleosomes Are Assembled
by a Replication-independent Pathway
Figure 4. Cid-GFP is deposited at centromeres by a replication-independent pathway. (A) Cells transfected with HS-H3GFP (open
bars) or HS-CidGFP (grey bars) were treated with aphidicolin to block DNA replication and then induced by heat-shock (no replication of DNA was seen in cells treated with aphidicolin, and then pulsed with dig-dUTP, data not shown). The number of cells showing
GFP localization in centromeres (of Cid-GFP) or in any part of the nucleus (H3-GFP) was compared with the number observed in induced, untreated cells. Localization of H3-GFP is replication dependent, but that of Cid-GFP is not. For each sample, 100–200 nuclei
were examined. (B) Cells transfected with heat-shock promoter-histone-GFP constructs were induced, and then loaded with dig-dUTP
to mark replicating DNA. In the merged images, sites of replicating DNA are in red, and protein localization is in green. Nuclei from
top to bottom are in early S phase, late S phase, and gap phase. H3GFP fusion protein localizes to replicating regions in S phase cells
and does not localize in gap phase cells. Cid-GFP fusion protein localizes to centromeres in all cell stages. The intensities of GFP signals
in each image are shown on the same absolute scale. Single optical sections are shown.
nucleotide analogues (Fig. 3; Henikoff et al., 2000), and
fail to localize in cells that are blocked by the replicationinhibiting drug aphidicolin (Fig. 4 A). Thus, the deposition
of these fusion proteins is coupled to the replication of the
underlying DNA, just as is the formation of conventional
nucleosomes (Adams and Kamakaka, 1999; Krude, 1999).
To determine whether the localization of Cid to centromeres is also coupled to replication, we transfected a heat
shock promoter-Cid-GFP fusion construct (HS-CidGFP)
into cells. Cells were then induced to express the fusion protein, or treated with aphidicolin to block DNA replication,
and then induced. Centromeric localization of Cid-GFP
occurred both in treated and untreated cells (Fig. 4 A),
demonstrating that the deposition of Cid need not be coupled to DNA replication. The replication independence of
Cid-GFP localization distinguishes it from that of the bulk
of conventional histones.
We further examined the localization of Cid-GFP to
centromeres when produced at different times in the cell
cycle. The deposition of histones onto DNA is predominantly limited to cells that are in S phase of the cell cycle
(Wu et al., 1986). We transfected cells with heat shock promoter-histone-GFP fusion constructs (HS-H3GFP or HSCidGFP). We then induced the construct and pulsed these
cells with nucleotide analogues to identify cells in S phase.
A pulse of H3-GFP protein localized in early and late replication patterns similar to the nucleotide analogue in the
same cells, and no detectable localization occurred in gapphase cells (Fig. 4 B, left). However, we found that newly
produced Cid-GFP protein localized to centromeres in
The Journal of Cell Biology, Volume 153, 2001
106
Figure 5. Deposition of CidGFP before mitosis. Cells
transfected with HS-H3GFP or
HS-CidGFP were induced and
prepared for cytology after
various chase times. We examined 50 metaphase figures
from each time point and
counted the number of labeled
mitotic figures. In the merged
images, Cid localization is in
red and histone-GFP in green.
Each image is a projection of
multiple sections through the
spread. (A) Mitotic chromosomes that show H3GFP labeling first appear 4 h after induction. H3GFP labels pericentric heterochromatin in these chromosomes, as expected for cells that
were in late S phase at the time of induction. (B) Metaphase spreads with Cid-GFP at centromeres appear earlier, 2 h after induction, indicating that these cells were in G2 phase when induced.
both S-phase and gap-phase cells with similar efficiencies
(Fig. 4 B, right). We confirmed that localization of CidGFP protein can occur in post-replicative cells, whereas
that of H3-GFP is strictly replication dependent, by examining metaphase chromosomes at various times after induction of transfected constructs. In cells transfected with
HS-H3GFP, occasional labeled metaphase figures first appear 4 h after induction and are labeled in heterochromatic segments (Fig. 5 A). The pattern of labeling and timing of these figures reflects the deposition of H3-GFP in
late S-phase cells and the shortest time required to
traverse the G2 phase of the cell cycle, and is consistent
with previous observations in Kc cell populations (Barigozzi et al., 1966; Dolfini et al., 1970). However, Cid-GFP
first appears at the centromeres in 48% of mitotic figures
within 2 h of induction: these must be from cells that were
induced during the G2 phase (Fig. 5 B). By 4 h s after induction, 100% of the mitotic figures from transfected cells
show centromeric labeling. We conclude that the Cid deposition pathway is present and active throughout the cell
cycle.
Analysis of centromeres in complex eukaryotes has been
hampered by the lack of sequence differences between the
centromere and flanking heterochromatin, and the repetitive nature of these regions (Sun et al., 1997; Willard,
1998). These sequence commonalities have led to the attribution of heterochromatic features to the centromere, including late replication. Our analysis demonstrates that
the replication of centromeres in Drosophila cells actually
precedes that of pericentromeric heterochromatin. We estimate that, on average, ⵑ500 kb of centromeric DNA is
replicated in the early S phase period. This size is in agreement with a genetically defined fully functional centromere in Drosophila (Murphy and Karpen, 1995), suggesting that the early replication domain corresponds to
the complete centromere. The early timing of its replica-
tion distinguishes the centromere from other repetitive sequences and rules out models for defining centromeres
that have invoked their very late replication (Dupraw,
1968; Csink and Henikoff, 1998). Early replication appears
to be a general feature of centromeres, as Saccharomyces
centromeres are known to replicate early in S phase (McCarroll and Fangman, 1988).
Our observations on the controlled assembly of conventional (H3-containing) and specialized (Cid-containing)
nucleosomes at replicating centromeres suggests that
chromatin assembly is a critical step in centromere maintenance. The cid promoter drives expression early in S phase
(Henikoff et al., 2000), and centromeres are replicating
during this time. Therefore, Cid synthesis and centromere
replication appear to be tightly coordinated. In Schizosaccharomyces yeast, the Cnp1 gene (encoding the centromeric SpCenpA histone) is also expressed early in S phase
(Takahashi et al., 2000), and we expect that a similar coordination with centromeric replication will be found.
At the time that Cid is being deposited, H3 deposition is
inhibited. It is striking that early replicating centromeres
are typically surrounded by late-replicating heterochromatin, and we suggest that inhibiting histone H3 incorporation at centromeres when they replicate is one function of
this juxtaposition. Inhibiting histone H3 incorporation at
centromeres requires the uncoupling of conventional
chromatin assembly and DNA replication. These two processes are thought to be linked by interactions between
replication machinery and the CAF1 chromatin assembly
factor (Krude, 1995; Verreault et al., 1996). Uncoupling
may be accomplished if histone H3 or some component of
its assembly machinery is excluded from the heterochromatic chromocenter early in S phase. Regions deficient in
histone H3 would then be incorporated into Cid-containing nucleosomes by a replication-independent pathway.
The observation that centromeric histone H3-like proteins from worms and yeast preferentially localize to fly or
human heterochromatin (Henikoff et al., 2000) suggests
that heterochromatin sequesters centromeric H3-like pro-
Ahmad and Henikoff Centromeres Are Specialized Replication Domains
107
Discussion
teins in general. Sequestering Cid in the heterochromatic
chromocenter would increase the local concentration of
Cid around centromeres and thereby promote Cid deposition. Centromeres in many organisms are typically surrounded by heterochromatin, and genetic evidence suggests that heterochromatin is important for centromere
function (Allshire et al., 1995). The centromeres of Saccharomyces chromosomes are the only known exception
to this rule, but in this organism centromeric activity is
conferred by a specific DNA sequence and associated
DNA-binding proteins (Meluh and Koshland, 1997). The
importance of heterochromatin for the function of complex centromeres is reinforced by the finding that a human
neocentromere shows M31 staining (a marker for mammalian heterochromatin), whereas the parental chromosomal region does not (Saffery et al., 2000). Perhaps the
exclusion of histone H3 during replication is one of the
prerequisites for “centromerization” (Choo, 2000), thus
necessitating that neocentromeres acquire heterochromatic proteins.
We expect that centromeres must be protected from
conventional nucleosome assembly pathways in all dividing cells, but heterochromatin may not always perform this
function. For example, distinct heterochromatin does not
form in the rapidly dividing nuclei of Drosophila syncytial
embryos (Hiraoka et al., 1993), and replication initiates
throughout the chromosomes simultaneously (Kriegstein
and Hogness, 1974). In these unusual nuclei, conventional
nucleosome assembly might be prevented by excluding
histone H3 from the apical edge of interphase nuclei,
where centromeres lie (Foe et al., 1993). Similarly, varying
local concentrations of proteins around nuclei have been
proposed to explain progression of the syncytial cell cycle
even though bulk cyclin levels are always high (Foe et al.,
1993). In later cycles, it appears to be most efficient to produce Cid when centromeres replicate.
It has been of great interest to understand how the location of the centromere is stably maintained in successive
cell divisions, because it does not appear that DNA sequence is responsible (Brown and Tyler-Smith, 1995;
Karpen and Allshire, 1997). Nucleosome particles form
the fundamental unit of chromatin, and so an attractive alternative to DNA sequence-based inheritance of centromere identity is that centromeric nucleosomes participate in centromere maintenance (Palmer et al., 1990).
Replication initiation appears to depend on chromatin
structure (Ofir et al., 1999; Stevenson and Gottschling,
1999) and we suggest that Cid-containing nucleosomes
predispose DNA to replicate early. This early replication
and the exclusion of histone H3 in heterochromatin would
preclude conventional chromatin assembly, thus allowing
the assembly of Cid-containing nucleosomes and ensuring
early replication again in the next cycle. This process
would maintain centromeres.
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Revised: 21 December 2000
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This work is dedicated to the memory of Doug Palmer. We thank members of the Henikoff lab for many valuable discussions, Jim Smothers for
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